Feed supplement material for use in aquaculture feed

ABSTRACT

The invention concerns a method of sustainably producing an aquaculture meat product by feeding a fish over its dietary cycles an aquaculture feed composition, said method comprising the step of formulating an aquaculture feed composition by replacing all or part of fish oil in the composition with a single microbial source of eicosapentaenoic acid (“EPA”) and docosahexaenoic acid (“DHA”). In a preferred embodiment, the microbial source comprising DHA and EPA derives from a microorganism/microbe of the genus  Schizochytrium  or  Thraustochytrium.

This application is the U.S. national phase of International ApplicationNo. PCT/EP2015/079417 filed Dec. 11, 2015 which designated the U.S. andclaims priority to EP Patent Application No. 14197713.2 filed Dec. 12,2014 and EP Patent Application No. 14020113.8 filed Dec. 18, 2014, theentire contents of each of which are hereby incorporated by reference.

This invention is in the field of aquaculture. More specifically, thisinvention pertains to a method of sustainably producing an aquaculturemeat product by feeding a fish over its dietary cycles an aquaculturefeed composition that includes at least a reduced amount of fish oil.

Aquaculture is a form of agriculture that involves the propagation,cultivation and marketing of aquatic animals and plants in a controlledenvironment. The aquaculture industry is currently the fastest growingfood production sector in the world. World aquaculture producesapproximately 60 million tons of seafood, which is worth more than $70billion (US) annually. Today, farmed fish accounts for approximately 50%of all fish consumed globally. This percentage is expected to increaseas a result of dwindling catches from capture fisheries in both marineand freshwater environments and increasing seafood consumption (i.e.,total and per capita). Today, species groups in aquaculture productioninclude, for example: carps and other cyprinids; oysters; clams, cocklesand arkshells; shrimps and prawns; salmons, trouts and smelts; mussels;tilapias and other cichlids; and scallops.

While some aquacultured species (e.g., Tilapia) can be fed on anentirely vegetarian diet, many others species are fed a carnivorousdiet. Typically, the feed for carnivorous fish comprises fishmeal andfish oil derived from wild caught species of small pelagic fish(predominantly anchovy, jack mackerel, blue whiting, capelin, sandeeland menhaden). These pelagic fish are processed into fishmeal and fishoil, with the final product often being either a pelleted or flakedfeed, depending on the size of the fish. The other components of theaquaculture feed composition may include vegetable protein, vitamins,minerals and pigment as required.

Marine fish oils have traditionally been used as the sole dietary lipidsource in commercial fish feed given their ready availability,competitive price and the abundance of essential fatty acids containedwithin this product. Additionally, fish oils readily supply essentialfatty acids which are required for regular growth, health, reproductionand bodily functions within fish. More specifically, all vertebratespecies, including fish, have a dietary requirement for both omega-6 andomega-3 polyunsaturated fatty acids [“PUFAs”]. Eicosapentaenoic acid[“EPA”; cis-5,8,11,14,17-eicosapentaenoic acid; omega-3] anddocosahexaenoic acid [“OHA”; cis-4,7,10,13,16,19-docosahexaenoic acid;22:6 omega-3] are required for fish growth and health and are oftenincorporated into commercial fish feeds via addition of fish oils.

It is estimated that aquaculture feed compositions currently use about87% of the global supply of fish oil as a lipid source. Since annualfish oil production has not increased beyond 1.5 million tons per year,the rapidly growing aquaculture industry cannot continue to rely onfinite stocks of marine pelagic fish as a supply of fish oil. Thus,there is great urgency to find and implement sustainable alternatives tofish oil that can keep pace with the growing global demand for fishproducts.

Many organizations recognize the limitations noted above with respect tofish oil availability and aquaculture sustainability. For example, inthe United States, the National Oceanic and Atmospheric Administrationis partnering with the Department of Agriculture in an Alternative FeedsInitiative to “ . . . identify alternative dietary ingredients that willreduce the amount of fishmeal and fish oil contained in aquaculturefeeds while maintaining the important human health benefits of farmedseafood”.

U.S. Pat. No. 7,932,077 suggests recombinantly engineered Yarrowialipolytica may be a useful addition to most animal feeds, includingaquaculture feeds, as a means to provide necessary omega-3 and/oromega-6 PUFAs and based on its unique protein:lipid:carbohydratecomposition, as well as unique complex carbohydrate profile (comprisingan approximate 1:4:4.6 ratio of mannan:beta-glucans:chitin).

U.S. Pat. Appl. Pub. No. 2007/0226814 discloses fish food containing atleast one biomass obtained from fermenting microorganisms wherein thebiomass contains at least 20% DHA relative to the total fatty acidcontent. Preferred microorganisms used as sources for DHA are organismsbelonging to the genus Stramenopiles.

If the growing aquaculture industry is to sustain its contribution toworld fish supplies while producing aquaculture meat products thatcontinue to provide health benefits for human consumption, then areduction in the use wild fish is needed along with the adoption of moreecologically-sound management practices of the world fish supply

SUMMARY OF THE INVENTION

In one embodiment, the invention concerns a method of sustainablyproducing an aquaculture meat product by feeding a fish over its dietarycycles an aquaculture feed composition, said method comprising the stepof formulating an aquaculture feed composition by replacing all or partof fish oil in the composition with a single microbial source ofeicosapentaenoic acid (“EPA”) and docosahexaenoic acid (“DHA”).

In a preferred example, the microbial source comprising DHA and EPA isproduced using a process based on the natural abilities of nativemicrobes of Schizochytrium species.

In a second embodiment, the invention concerns a method of sustainablyproducing an aquaculture meat product by feeding a fish over its dietarycycles an aquaculture feed composition wherein the aquaculture feedcomposition comprises a total amount of EPA and DHA derived from saidmicrobial source that is at least about 0.8% measured as a weightpercent of the aquaculture feed composition.

In a third embodiment, the invention concerns a method of sustainablyproducing an aquaculture meat product by feeding a fish over its dietarycycles an aquaculture feed composition with a microbial oil source ofEPA and DHA, wherein the microbial oil is provided in a form selectedfrom the group consisting of: biomass, processed biomass, partiallypurified oil and purified oil, any of which is obtained from onemicrobe.

In a fourth embodiment, the invention concerns a feed additivecomposition for fish feed products, said additive composition comprisesa single microbial source of eicosapentaenoic acid (“EPA”) anddocosahexaenoic acid (“DHA”).

In a fifth embodiment, the invention concerns aquaculture feed with amicrobial additive composition containing EPA and DHA, wherein themicrobial additive is obtained from one single microbe.

In a sixth embodiment, the invention concerns a method of sustainablyproducing an aquaculture meat product by feeding a fish over its dietarycycles an aquaculture feed composition, said method comprising the stepof formulating an aquaculture feed composition by replacing all or partof fish oil in the composition with a single microbial source ofeicosapentaenoic acid (“EPA”) and docosahexaenoic acid (“DHA”), whereinsaid microbe is a transgenic microbe genetically engineered for theproduction of polyunsaturated fatty acid containing microbial oilcomprising EPA and DHA.

Preferably, the transgenic microbe is a microorganism of the orderThraustochytriales.

DETAILED DESCRIPTION

In this disclosure, a number of terms and abbreviations are used. Thefollowing definitions are provided:

“Polyunsaturated fatty acid(s)” is abbreviated as “PUFA(s)”.

“Triacylglycerols” are abbreviated as “TAGs”.

“Total fatty acids” are abbreviated as “TFAs”.

“Fatty acid methyl esters” are abbreviated as “FAMEs”.

“Dry cell weight” is abbreviated as “DCW”.

As used herein the term “invention” or “present invention” is intendedto refer to all aspects and embodiments of the invention as described inthe claims and specification herein and should not be read so as to belimited to any particular embodiment or aspect.

The term “dietary cycles” of a fish refers to periods or stages ofgrowth (i.e., growth stages) during which fish are fed a diet, oraquaculture feed, during aquaculture production. An example of dietarycycles for Atlantic Salmon is set forth in Table 1 below where there aresix stages corresponding to the noted starting and ending weights. Thedietary cycles in terms of number of stages, as well as starting andending weights of fish for each stage, may vary for different types offish and/or for different aquaculture practices.

TABLE 1 Exemplary Dietary Cycles or Stages of Fish Growth Stage 1 2 3 45 6 Starting Weight (g) 100 250 800 1500 2500 3500 Ending Weight (g) 250800 1500 2500 3500 4500

The terms “aquaculture feed composition”, “aquaculture feedformulation”, “aquaculture feed” and “aquafeed” are used interchangeablyherein. They refer to manufactured or artificial diets (i.e., formulatedfeeds) to supplement or to replace natural feeds in the aquacultureindustry. These prepared foods are most commonly produced in flake,pellet or tablet form. Typically, an aquaculture feed composition refersto artificially compounded feeds that are useful for farmed finfish andcrustaceans (i.e., both lower-value staple food fish species [e.g.,freshwater finfish such as carp, tilapia and catfish] and higher-valuecash crop species for luxury or niche markets [e.g., mainly marine anddiadromous species such as shrimp, salmon, trout, yellowtail, seabass,seabream and grouper]). These formulated feeds are composed ofingredients in various proportions complementing each other to form anutritionally complete diet for the aquacultured species. An aquaculturefeed composition is used in the production of an “aquaculture product”,wherein the product is a harvestable aquacultured species (e.g.,finfish, crustaceans), which is often sold for human consumption. Forexample, salmon are intensively produced in aquaculture and thus areaquaculture products.

The term “aquaculture meat product” refers to food products intended forhuman consumption comprising at least a portion of meat from anaquaculture product as defined above. An aquaculture meat product maybe, for example, a whole fish or a filet cut from a fish, each of whichmay be consumed as food.

“Eicosapentaenoic acid” [“EPA”] is the common name foreis-5,8,11,14,17-eicosapentaenoic acid. This fatty acid is a 20:5omega-3 fatty acid. The term EPA as used in the present disclosure willrefer to the acid or derivatives of the acid (e.g., glycerides, esters,phospholipids, amides, lactones, salts or the like) unless specificallymentioned otherwise.

“Docosahexaenoic acid” [“DHA”] is the common name foreis-4,7,10,13,16,19-docosahexaenoic acid. This fatty acid is a 22:6omega-3 fatty acid. The term DHA as used in the present disclosure willrefer to the acid or derivatives of the acid (e.g., glycerides, esters,phospholipids, amides, lactones, salts or the like) unless specificallymentioned otherwise.

As used herein the term “additive composition” refers to materialderived from a microbial source which is provided in a form selectedfrom the group consisting of: biomass, processed biomass, partiallypurified oil and purified oil, any of which is obtained from one singlemicrobe.

As used herein the term “biomass” refers to microbial cellular material.Biomass may be produced naturally, or may be produced from thefermentation of a native host or a mutant strain or a recombinantproduction host. The biomass may be in the form of whole cells, wholecell-lysates, homogenized cells, partially hydrolyzed cellular material,and/or partially purified cellular material (e.g., microbially producedoil). The term “processed biomass” refers to biomass that has beensubjected to additional processing such as drying, pasteurization,disruption, etc., each of which is discussed in greater detail below.

The term “lipids” refer to any fat-soluble (i.e., lipophilic), naturallyoccurring molecule. A general overview of lipids is provided in U.S.Pat. Appl. Pub. No. 2009-0093543-A1. The term “oil” refers to a lipidsubstance that is liquid at 25° C. and usually polyunsaturated.

The term “extracted oil” refers to oil that has been separated fromcellular materials, such as the microorganism in which the oil wassynthesized. Extracted oils are obtained through a wide variety ofmethods, the simplest of which involves physical means alone. Forexample, mechanical crushing using various press configurations (e.g.,screw, expeller, piston, bead beaters, etc.) can separate oil fromcellular materials. Alternatively, oil extraction can occur viatreatment with various organic solvents (e.g., hexane), via enzymaticextraction, via osmotic shock, via ultrasonic extraction, viasupercritical fluid extraction (e.g., CO₂ extraction), viasaponification and via combinations of these methods. An extracted oilmay be further purified or concentrated.

“Fish oil” refers to oil derived from the tissues of an oily fish.Examples of oily fish include, but are not limited to: menhaden,anchovy, herring, capelin, cod and the like. Fish oil is a typicalcomponent of feed used in aquaculture.

“Vegetable oil” refers to any edible oil obtained from a plant.Typically plant oil is extracted from seed or grain of a plant. The term“triacylglycerols” [“TAGs”] refers to neutral lipids composed of threefatty acyl residues esterified to a glycerol molecule.

TAGs can contain long chain PUFAs and saturated fatty acids, as well asshorter chain saturated and unsaturated fatty acids. “Neutral lipids”refer to those lipids commonly found in cells in lipid bodies as storagefats and are so called because at cellular pH, the lipids bear nocharged groups. Generally, they are completely non-polar with noaffinity for water. Neutral lipids generally refer to mono-, di-, and/ortriesters of glycerol with fatty acids, also called monoacylglycerol,diacylglycerol or triacylglycerol, respectively, or collectively,acylglycerols. A hydrolysis reaction must occur to release free fattyacids from acylglycerols.

The term “total fatty acids” [“TFAs”] herein refers to the sum of allcellular fatty acids that can be derivatized to fatty acid methyl esters[“FAMEs”] by the base transesterification method (as known in the art)in a given sample, which may be biomass or oil, for example. Thus, totalfatty acids include fatty acids from neutral lipid fractions (includingdiacylglycerols, monoacylglycerols and TAGs) and from polar lipidfractions (including, e.g., the phosphatidylcholine andphosphatidylethanolamine fractions) but not free fatty acids.

The term “total lipid content” of cells is a measure of TFAs as apercent of the dry cell weight [“DeW”]′ although total lipid content canbe approximated as a measure of FAMEs as a percent of the DeW [“FAMEs %DeW”]. Thus, total lipid content [“TFAs % DeW”] is equivalent to, e.g.,milligrams of total fatty acids per 100 milligrams of DeW.

The concentration of a fatty acid in the total lipid is expressed hereinas a weight percent of TFAs (% TFAs), e.g., milligrams of the givenfatty acid per 100 milligrams of TFAs. Unless otherwise specificallystated in the disclosure herein, reference to the percent of a givenfatty acid with respect to total lipids is equivalent to concentrationof the fatty acid as % TFAs (e.g., % EPA of total lipids is equivalentto EPA % TFAs).

In some cases, it is useful to express the content of a given fattyacid(s) in a cell as its weight percent of the dry cell weight (% DCW).Thus, for example, eicosapentaenoic acid % DCW would be determinedaccording to the following formula: (eicosapentaenoic acid % TFAs) (TFAs% DCW)]/100. The content of a given fatty acid(s) in a cell as itsweight percent of the dry cell weight (% DCW) can be approximated,however, as: (eicosapentaenoic acid % TFAs)*(FAMEs % DCW)]/100.

The terms “lipid profile” and “lipid composition” are interchangeableand refer to the amount of individual fatty acids contained in aparticular lipid fraction, such as in the total lipid or the oil,wherein the amount is expressed as a weight percent of TFAs. The sum ofeach individual fatty acid present in the mixture should be 100.

The term “blended oil” refers to an oil that is obtained by admixing, orblending, the extracted oil described herein with any combination of, orindividual, oil to obtain a desired composition. Thus, for example,types of oils from different microbes can be mixed together to obtain adesired PUFA composition. Alternatively, or additionally, thePUFA-containing oils disclosed herein can be blended with fish oil,vegetable oil or a mixture of both to obtain a desired composition.

The term “fatty acids” refers to long chain aliphatic acids (alkanoicacids) of varying chain lengths, from about C12 to C22, although bothlonger and shorter chain-length acids are known. The predominant chainlengths are between C16 and C22. The structure of a fatty acid isrepresented by a simple notation system of “X:Y”, where X is the totalnumber of carbon [“C”] atoms in the particular fatty acid and Y is thenumber of double bonds. Additional details concerning thedifferentiation between “saturated fatty acids” versus “unsaturatedfatty acids”, “monounsaturated fatty acids” versus “polyunsaturatedfatty acids” [“PUFAs”], and “omega-6 fatty acids” [“00-6” or “n-6”]versus “omega-3 fatty acids” [“00-3” or “n-3”] are provided in U.S. Pat.No. 7,238,482, which is hereby incorporated herein by reference.

“Fish meal” refers to a protein source for aquaculture feedcompositions. Fish meals are typically either produced from fisherywastes associated with the processing of fish for human consumption(e.g., salmon, tuna) or produced from specific fish (i.e., herring,menhaden) which are harvested solely for the purpose of producing fishmeal.

Aquaculture is the practice of farming aquatic animals and plants. Itinvolves cultivating an aquatic product (e.g., freshwater and saltwateranimals) under controlled conditions. It involves growing and harvestingfish, shellfish, and aquatic plants in fresh, brackish or salt water.

Organisms grown in aquaculture may include fish and crustaceans.Crustaceans are, for example, lobsters, crabs, shrimp, prawns andcrayfish. The farming of finfish is the most common form of aquaculture.

It involves raising fish commercially in tanks, ponds, or oceanenclosures, usually for food. A facility that releases juvenile fishinto the wild for recreational fishing or to supplement a species'natural numbers is generally referred to as a fish hatchery.Particularly of interest are fish of the salmonid group, for example,cherry salmon (Oncorhynchus rnasou), Chinook salmon (O. tshawytscha),chum salmon (O. keta), coho salmon (O. kisutch), pink salmon (O.gorbuscha), sockeye salmon (O. nerka) and Atlantic salmon (Salmo salar).Other finfish of interest for aquaculture include, but are not limitedto, various trout, as well as whitefish such as tilapia (includingvarious species of Oreochromis, Sarotherodon, and Tilapia), sea bass,catfish (order Siluri-formes), bigeye tuna (Thunnus obesus), carp(family Cyprinidae) and cod (Gadus).

Aquaculture typically requires a prepared aquaculture feed compositionto meet dietary requirements of the cultured animals. Dietaryrequirements of different aquaculture species vary, as do the dietaryrequirements of a single species during different stages of growth.Thus, tremendous research is invested towards optimizing eachaquaculture feed composition for each stage of growth of a culturedorganism.

Aquaculture feed compositions are composed of micro and macrocomponents. In general, all components, which are used at levels of morethan 1%, are considered as macro components. Feed ingredients used atlevels of less than 1% are micro components. They are premixed toachieve a homogeneous distribution of the micro components in thecomplete feed. Both macro and micro ingredients are subdivided intocomponents with nutritional functions and technical functions.

Components with technical functions improve the physical quality of theaquaculture feed composition or its appearance.

Macro components with nutritional functions provide aquatic animals withprotein and energy required for growth and performance. With respect tofish, the aquaculture feed composition should ideally provide the fishwith: 1) fats, which serve as a source of fatty acids for energy(especially for heart and skeletal muscles); and, 2) amino acids, whichserve as building blocks of proteins. Fats also assist in vitaminabsorption; for example, vitamins A, D, E and K are fat-soluble or canonly be digested, absorbed, and transported in conjunction with fats.Carbohydrates, typically of plant origin (e.g., wheat, sunflower meal,corn gluten, soybean meal), are also often included in the feedcompositions, although carbohydrates are not a superior energy sourcefor fish over protein or fat.

Fats are typically provided via incorporation of fish meals (whichcontain a minor amount of fish oil) and fish oils into the aquaculturefeed compositions. Extracted oils that may be used in aquaculture feedcompositions include fish oils (e.g., from the oily fish menhaden,anchovy, herring, capelin and cod liver), and vegetable oil (e.g., fromsoybeans, rapeseeds, sunflower seeds and flax seeds). Typically, fishoil is the preferred oil, because it contains the long chain omega-3polyunsaturated fatty acids [“PUFAs”], EPA and DHA; in contrast,vegetable oils do not provide a source of EPA and/or DHA. These PUFAsare needed for growth and health of most aquaculture products. A typicalaquaculture feed composition will comprise from about 15-30% of oil(e.g., fish, vegetable, etc.), measured as a weight percent of theaquaculture feed composition.

The amount of EPA (as a percent of total fatty acids [“% TFAs”]) and DHA% TFAs provided in typical fish oils varies, as does the ratio of EPA toDHA. Typical values are summarized in Table 2, based on the work ofTurchini, Torstensen and Ng (Reviews in Aquaculture 1:10-57 (2009)):

TABLE 2 Typical EPA and DHA Content in Various Fish Oils Fish Oil EPADHA EPA:DHA Ratio Anchovy oil  17% 8.8% 1.93:1 Capelin oil 4.6% 3.0%1.53:1 Menhaden oil  11% 9.7% 1.21:1 Herring oil 8.4% 4.9% 1.71:1

The protein supplied in aquaculture feed compositions can be of plant oranimal origin. For example, protein of animal origin can be from marineanimals (e.g., fish meal, fish oil, fish protein, krill meal, musselmeal, shrimp peel, squid meal, squid oil, etc.) or land animals (e.g.,blood meal, egg powder, liver meal, meat meal, meat and bone meal,silkworm, pupae meal, whey powder, etc.). Protein of plant origin caninclude soybean meal, corn gluten meal, wheat gluten, cottonseed meal,canola meal, sunflower meal, rice and the like.

The technical functions of macro components can be overlapping as, forexample, wheat gluten may be used as a pelleting aid and for its proteincontent, which has a relatively high nutritional value. There can alsobe mentioned guar gum and wheat flour.

Micro components include feed additives such as vitamins, traceminerals, feed antibiotics and other biologicals. Minerals used atlevels of less than 100 mg/kg (100 ppm) are considered as micro mineralsor trace minerals.

Micro components with nutritional functions are all biologicals andtrace minerals. They are involved in biological processes and are neededfor good health and high performance. There can be mentioned vitaminssuch as vitamins A, E, K3, D3, B1, B3, B6, B12, C, biotin, folic acid,panthothenic acid, nicotinic acid, choline chloride, inositol andpara-amino-benzoic acid. There can be mentioned minerals such as saltsof calcium, cobalt, copper, iron, magnesium, phosphorus, potassium,selenium and zinc. Other components may include, but are not limited to,antioxidants, beta-glucans, bile salt, cholesterol, enzymes, monosodiumglutamate, carotenoids, etc.

The technical functions of micro ingredients are mainly related topelleting, detoxifying, mold prevention, antioxidation, etc.

In aquaculture, typically fish are fed in different dietary cycles asthey grow. For example, Atlantic salmon may be fed in six differentdietary cycles while growing from 100 grams to 4 kilograms as shown inTable 1 above. The weights of fish of different dietary cycles may varydepending on the type of fish and/or the aquaculture practice used.

In the second aspect, the aquaculture feed composition may comprise atotal amount of EPA and DHA derived from a single microbial source thatis at least about 0.8%, measured as weight percent of the aquaculturefeed composition. This amount (i.e., 0.8%) is typically an appropriateminimal concentration that is suitable to support the growth of avariety of animals grown in aquaculture, and particularly is suitablefor inclusion in the diets of salmonid fish.

As previously discussed, the highest EPA:DHA ratio in fish oil (i.e.,anchovy oil) was 1.93:1 (Turchini, Torstensen and Ng, supra) and thelowest EPA:DHA ratio in fish oil was 1.21:1. Thus, it is believed thatno commercially available aquaculture feed composition has been producedhaving an EPA:DHA ratio greater than 1.93:1 or lower than 1.21:1.

To achieve an EPA:DHA ratio greater than 2:1 or lower than 1.2:1, asdescribed herein, an alternate source of EPA and DHA is required.

In one example, the aquaculture feed compositions of the presentinvention comprise one source of DHA and EPA, wherein the ratio ofEPA:DHA is greater than 2:1 based on the individual concentrations ofEPA and DHA, each measured as a weight percent of total fatty acids inthe microbial source or in the aquaculture feed composition.

In another example, the aquaculture feed compositions of the presentinvention comprise one source of DHA and EPA, wherein the ratio ofEPA:DHA is lower than 1:1, preferably between 0.2:1 and 1:1 based on theindividual concentrations of EPA and DHA, each measured as a weightpercent of total fatty acids in the microbial source or in theaquaculture feed composition.

Most processes to make an aquaculture feed composition of the inventionwill begin with a microbial fermentation, wherein a particularmicroorganism is cultured under conditions that permit growth andproduction of microbial oils comprising EPA and DHA. At an appropriatetime, the microbial cells are harvested from the fermentation vessel.This microbial biomass may be mechanically processed using variousmeans, such as dewatering, drying, mechanical disruption, pelletization,etc. Then, the biomass (or extracted oil therefrom) is used as aningredient in an aquaculture feed (preferably as a substitute for atleast a portion of the fish oil used in standard aquaculture feedcompositions). The aquaculture feed is then fed to aquatic animals overa portion of their lifetime, such that EPA and DHA from the aquaculturefeed accumulate in the aquatic animals. Upon harvesting, the resultingaquaculture meat product will thereby comprise a ratio of EPA/DHA thatis equal to or greater than 2:1 or equal or lower than 1:1. Each ofthese aspects will be discussed in further detail below.

Microbial oils comprising EPA and DHA according to the present inventionmay be provided in a variety of forms for use in the aquaculture feedcompositions herein, wherein the oil is typically contained withinmicrobial biomass or processed biomass, or the oil is partially purifiedor purified oil. In most cases, it will be most cost effective toincorporate microbial biomass or processed biomass into the aquaculturefeed composition, as opposed to the microbial oil (in partial orpurified form); however, these economics should not be considered as alimitation herein.

The microorganism according to the present invention is an algae, fungior yeast. Preferred microbes are Thraustochytrids which aremicroorganisms of the order Thraustochytriales. Thraustochytrids includemembers of the genus Schizochytrium and Thraustochytrium and have beenrecognized as an alternative source of omega-3 fatty acids, includingDHA and EPA. See U.S. Pat. No. 5,130,242.

In a preferred embodiment the microorganism is a mutant strain of thespecies Schizochytrium. Schizochytrium strains are natural sources ofPUFAs such as DHA and can be optimized by mutagenesis to be used asmicrobial source according to the present invention.

DHA and EPA producing Schizochytrium strains can be obtained byconsecutive mutagenesis followed by suitable selection of mutant strainswhich demonstrate superior EPA and DHA production and a specific EPA:DHAratio. Starting wild type strains include those on deposit with thevarious culture collections throughout the world, e.g. the ATCC and theCentraalbureau voor Schimmelcultures (CBS). Typically it is necessary toperform two or more consecutive rounds of mutagenesis to obtaindesirable mutant strains.

Any chemical or nonchemical (e.g. ultraviolet (UV) radiation) agentcapable of inducing genetic change to the yeast cell can be used as themutagen. These agents can be used alone or in combination with oneanother, and the chemical agents can be used neat or with a solvent.

For example, a strain can be mutated and selected such that it producesEPA and DHA in amounts to be commercially viable and with a EPA:DHAratio equal to or greater than 2:1 and may with a ratio of at leastabout 2.2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, 5.5:1, 6:1, 6.5:1, 7:1,7.5:1, 8:1, 8.5:1, 9:1, 9.5:1, or 10:1 or higher. Alternatively thestrain can be mutated and selected such that it produces EPA and DHA inamounts to be commercially viable and with a EPA:DHA ratio equal to orlower than 1:1 and may with a ratio of about 0.9:1, 0.8:1, 0.7:1, 0.6:1,0.5:1, 0.4:1, 0.3:1, 0:2:1 or lower.

Alternately, the microbial source according the invention can beproduced by microbes genetically transformed for the production of thePUFAs. Optionally the microorganism may be engineered for production ofDHA and EPA by expressing appropriate heterologous genes encoding forexample desaturases and elongases of either the delta-6desaturase/delta-6 elongase pathway or the delta-9 elongase/delta-8desaturase pathway in the host organism.

Heterologous genes in expression cassettes are typically integrated intothe host cell genome. The particular gene(s) included within aparticular expression cassette depend on the host organism, its PUFAprofile and/or desaturase/elongase profile, the availability ofsubstrate and the desired end product(s). A PUFA polyketide synthase[“PKS”] system that produces EPA, such as that found in e.g., Shewanellaputrefaciens (U.S. Pat. No. 6,140,486), Shewanella olleyana (U.S. Pat.No. 7,217,856), Shewanella japonica (U.S. Pat. No. 7,217,856) and Vibriomarinus (U.S. Pat. No. 6,140,486), could also be introduced into asuitable DHA producing microbe to enable EPA and DHA production. Hostorganisms with other PKS systems that natively produce DHA could also beengineered to enable production of a suitable combination of the PUFAsto yield an EPA:DHA ratio of greater than 2:1 or lower than 1:1.

One skilled in the art is familiar with the considerations andtechniques necessary to introduce one or more expression cassettesencoding appropriate enzymes for EPA and DHA biosynthesis into amicrobial host organism of choice, and numerous teachings are providedin the literature to one of skill. Microbial oils comprising EPA and DHAfrom these genetically engineered organisms may also be suitable for usein the aquaculture feed compositions herein, wherein the oil may becontained within the microbial biomass or processed biomass, or the oilmay be partially purified or purified oil.

Typical species of microorganisms useful for the present invention aredeposited under ATCC Accession No. PTA-10208, PTA-10209, PTA-10210, orPTA-10211, PTA-10212, PTA-10213, PTA-10214, PTA-10215.

In some embodiments, the invention is directed to an isolatedmicroorganism having the characteristics of the species deposited underATCC Accession No. PTA-10212 or a strain derived therefrom. Thecharacteristics of the species deposited under ATCC Accession No.PTA-10212 can include its growth and phenotypic properties (examples ofphenotypic properties include morphological and reproductiveproperties), its physical and chemical properties (such as dry weightsand lipid profiles), its gene sequences, and combinations thereof, inwhich the characteristics distinguish the species over previouslyidentified species. In some embodiments, the invention is directed to anisolated microorganism having the characteristics of the speciesdeposited under ATCC Accession No. PTA-10212, wherein thecharacteristics include an 18s rRNA comprising the polynucleotidesequence of SEQ ID NO1 or a polynucleotide sequence having at least 94%,95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO1, the morphological andreproductive properties of the species deposited under ATCC AccessionNo. PTA-10212, and the fatty acid profiles of the species depositedunder ATCC Accession No. PTA-10212.

In further embodiments, the mutant strain is a strain deposited underATCC Accession No. PTA-10213, PTA-10214, or PTA-10215. Themicroorganisms associated with ATCC Accession Nos. PTA-10213, PTA-10214,and PTA-10215 were deposited under the Budapest Treaty on Jul. 14, 2009at the American Type Culture Collection, Patent Depository, 10801University Boulevard, Manassas, Va. 20110-2209.

In some embodiments, the invention is directed to an isolatedmicroorganism of the species deposited under ATCC Accession No.PTA-10208. The isolated microorganism associated with ATCC Accession No.PTA-10208 is also known herein as Schizochytrium sp. ATCC PTA-10208. Themicroorganism associated with ATCC Accession No. PTA-10208 was depositedunder the Budapest Treaty on Jul. 14, 2009 at the American Type CultureCollection, Patent Depository, 10801 University Boulevard, Manassas, Va.20110-2209.

In some embodiments, the invention is directed to a mutant strain of themicroorganism deposited under ATCC Accession No. PTA-10208. In furtherembodiments, the mutant strain is a strain deposited under ATCCAccession No. PTA-10209, PTA-10210, or PTA-10211. The microorganismsassociated with ATCC Accession Nos. PTA-10209, PTA-10210, and PTA-10211were deposited under the Budapest Treaty on Sep. 25, 2009 at theAmerican Type Culture Collection, Patent Depository, 10801 UniversityBoulevard, Manassas, Va. 20110-2209.

A microbe according to the present invention may be cultured and grownin a fermentation medium under conditions whereby the PUFAs are producedby the microorganism. Typically, the microorganism is fed with a carbonand nitrogen source, along with a number of additional chemicals orsubstances that allow growth of the microorganism and/or production ofEPA and DHA. The fermentation conditions will depend on themicroorganism used and may be optimized for a high content of thedesired PUFA(s) in the resulting biomass.

In general, media conditions may be optimized by modifying the type andamount of carbon source, the type and amount of nitrogen source, thecarbon-to-nitrogen ratio, the amount of different mineral ions, theoxygen level, growth temperature, pH, length of the biomass productionphase, length of the oil accumulation phase and the time and method ofcell harvest.

When the desired amount of EPA and DHA has been produced by themicroorganism(s), the fermentation medium may be treated to obtainmicrobial biomass comprising the PUFA(s). For example, the fermentationmedium may be filtered or otherwise treated to remove at least part ofthe aqueous component. The fermentation medium and/or the microbialbiomass may be further processed, for example the microbial biomass maybe pasteurized or treated via other means to reduce the activity ofendogenous microbial enzymes that can harm the microbial oil and/orPUFAs. The microbial biomass may be subjected to drying (e.g., to adesired water content) or a means of mechanical disruption (e.g., viaphysical means such as bead beaters, screw extrusion, etc. to providegreater accessibility to the cell contents), or a combination of these.The microbial biomass may be granulated or pelletized for ease ofhandling. Thus, microbial biomass obtained from any of the meansdescribed above may be used as a source of microbial oil comprising EPAand DHA. This source of microbial oil may then be used as an ingredientin the aquaculture feed compositions.

In the first examples of the present invention, aquaculture meatproducts comprising EPA and DHA in a ratio that is equal to or greaterthan 2:1, based on the concentration of each of EPA and DHA in theaquaculture meat product, are sustainably produced. The ratio ofconcentration of each of EPA to DHA may be equal to or greater than 2:1,2.1:1, 2.2:1, 2.3:1, 2.4:1, 2.5:1, 2.6:1, 2.7:1, 2.8:1, 2.9:1, 3:1,3.5:1, 4:1, 4.5:1, 5:1, 5.5:1, 6:1, 6.5:1, 7:1, 7.5:1, 8:1, 8.5:1, 9:1,9.5:1, or 10:1 or higher.

In the second examples of the present invention, aquaculture meatproducts comprising EPA and DHA in a ratio that is equal to or lowerthan 1:1, based on the concentration of each of EPA and DHA in theaquaculture meat product, are sustainably produced. The ratio ofconcentration of each of EPA to DHA may be equal to or lower than 1:1,0.9:1, 0.8:1, 0.7:1, 0.6:1, 0.5:1, 0.4:1, 0.3:1, 0:2:1 or lower.

A preferred example of a microbial oil according to the invention is anoil from Schizochytrium containing

-   -   at least 40% w/w DHA & EPA, preferably about 50% w/w DHA & EPA,    -   an EPA:DHA ratio of about 0.2:1 to 1:1, preferably 0.4:1 to        0.8:1, and    -   at least one antioxidant which is added to provide stability.

Microbial oil as described and obtained from any of the means describedabove may be used as a single source of EPA and DHA for use inaquaculture feed compositions that are fed to aquaculture animals toproduce aquaculture meat products having an EPA:DHA ratio equal to orgreater than 2:1 or equal to or less than 1:1.

Aquaculture meat products obtained using the method of the invention mayfurther comprise a total amount of EPA and DHA that is at least about0.5% as a weight percent of the aquaculture meat product. This amount isan amount that typically is present in aquaculture meat products.

A total amount of EPA and DHA that is at least about 0.5% as weightpercent of an aquaculture meat product may be obtained by feedingaquaculture animals with an aquaculture feed composition having a sum ofEPA plus DHA that is typically at least about 1.6% of the aquaculturefeed composition by weight.

Based on the disclosure herein, it will be clear that renewablealternatives to fish oil can be utilized, as a means to sustainablyproduce aquaculture feed compositions over the dietary cycles of a fish.

Having generally described this invention, a further understanding canbe obtained by reference to the examples provided herein. These examplesare for purposes of illustration only and are not intended to belimiting.

Example 1 Growth Characteristics of the Isolated Microorganism DepositedUnder ATCC Accession No. PTA-10212

The isolated microorganism deposited under ATCC Accession No. PTA-10212was examined for growth characteristics in individual fermentation runs,as described below. Typical media and cultivation conditions are shownin Table 3.

TABLE 3 PTA-10212 Vessel Media Ingredient concentration ranges Na₂S0₄g/L 31.0 0-50, 15-45, or 25-35 NaCl g/L 0.625 0-25, 0.1-10, or 0.5-5 KClg/L 1.0 0-5, 0.25-3, or 0.5-2 MgS0₄*7H₂0 g/L 5.0 0-10, 2-8, or 3-6(NH₄)₂S0₄ g/L 0.44 0-10, 0.25-5, or 0.05-3 MSG*1H20 g/L 6.0 0-10, 4-8,01-5-7 CaCl₂ g/L 0.29 0.1-5, 0.15-3, or 0.2-1 T 154 (yeast extract) g/L6.0 0-20, 0.1-10, or 1-7 KH₂P0₄ g/L 0.8 0.1-10, 0.5-5, or 0.6-1.8 Postautoclave (Metals) Citric acid mg/L 3.5 0.1-5000, 10-3000, or 3-2500FeSO₄*7H₂O mg/L 10.30 0.1-100, 1-50, or 5-25 MnCl₂*4H₂O mg/L 3.100.1-100, 1-50, or 2-25 ZnS0₄*7H₂O mg/L 3.10 0.01-100, 1-50, or 2-25CoCl₂*6H₂O mg/L 0.04 0-1, 0.001-0.1, or 0.01-0.1 Na₂MoO₄*2H₂O mg/L 0.040.001-1, 0.005-0.5, or 0.01-0.1 CuSO₄*5H₂O mg/L 2.07 0.1-100, 0.5-50, or1-25 NiSO₄*6H₂O mg/L 2.07 0.1-100, 0.5-50, or 1-25 Post autoclave(Vitamins) Thiamine mg/L 9.75 0.1-100, 1-50, or 5-25 Vitamin B 12 mg/L0.16 0.01-100, 0.05-5, or 0.1-1 Ca[1/2]-pantothenate mg/L 2.06 0.1-100,0.1-50, or 1-10 Biotin mg/L 3.21 0.1-100, 0.1-50, or 1-10 Post autoclave(Carbon) Glycerol g/L 30.0 5-150, 10-100, or 20-50 Nitrogen Feed:MSG*1H₂O g/L 17 0-150, 10-100, or 15-50

Typical cultivation conditions would include the following:

-   pH 6.5-9.5, about 6.5-about 8.0, or about 6.8-about 7.8;-   temperature: 15-30 degrees Celsius, about 18-about 28 degrees    Celsius, or about 21 to about 23 degrees Celsius;-   dissolved oxygen: 0.1-about 100% saturation, about 5-about 50%    saturation, or about 10-about 30% saturation; and/or-   glycerol controlled @: 5-about 50 g/L, about 10-about 40 g/L, or    about 15-about 35 g/L.

In carbon (glycerol) and nitrogen-fed cultures with 1000 ppm CI at 22.5°C. with 20% dissolved oxygen at pH 7.3, PTA-10212 produced a dry cellweight of 26.2 g/L after 138 hours of culture in a 10 L fermentorvolume. The lipid yield was 7.9 g/L; the omega-3 yield was 5.3 g/L; theEPA yield was 3.3 g/L and the DHA yield was 1.8 g/L. The fatty acidcontent was 30.3% by weight; the EPA content was 41.4% of fatty acidmethyl esters (FAME); and the DHA content was 26.2% of FAME. The lipidproductivity was 1.38 g/L/day, and the omega-3 productivity was 0.92g/L/day under these conditions, with 0.57 g/L/day EPA productivity and0.31 g/L/day DHA productivity.

In carbon (glycerol) and nitrogen-fed cultures with 1000 ppm CI at 22.5°C. with 20% dissolved oxygen at pH 7.3, PTA-10212 produced a dry cellweight of 38.4 g/L after 189 hours of culture in a 10 L fermentorvolume. The lipid yield was 18 g/L; the omega-3 yield was 12 g/L; theEPA yield was 5 g/L and the DHA yield was 6.8 g/L. The fatty acidcontent was 45% by weight; the EPA content was 27.8% of FAME; and theDHA content was 37.9% of FAME. The lipid productivity was 2.3 g/L/day,and the omega-3 productivity was 1.5 g/L/day under these conditions,with 0.63 g/L/day EPA productivity and 0.86 g/L/day DHA productivity.

In carbon (glycerol) and nitrogen-fed cultures with 1000 ppm CI at 22.5°C. with 20% dissolved oxygen at pH 6.8-7.7, PTA-10212 produced a drycell weight of 13 g/L after 189 hours of culture in a 10 L fermentorvolume. The lipid yield was 5.6 g/L; the omega-3 yield was 3.5 g/L; theEPA yield was 1.55 g/L and the DHA yield was 1.9 g/L. The fatty acidcontent was 38% by weight; the EPA content was 29.5% of FAME; and theDHA content was 36% of FAME. The lipid productivity was 0.67 g/L/day,and the omega-3 productivity was 0.4 g/L/day under these conditions,with 0.20 g/L/day EPA productivity and 0.24 g/L/day DHA productivity.

In carbon (glycerol) and nitrogen-fed cultures with 1000 ppm CI at22.5-28.5° C. with 20% dissolved oxygen at pH 6.6-7.2, PTA-10212produced a dry cell weight of 36.7 g/L-48.7 g/L after 191 hours ofculture in a 10 L fermentor volume. The lipid yield was 15.2 g/L-25.3g/L; the omega-3 yield was 9.3 g/L-13.8 g/L; the EPA yield was 2.5g/L-3.3 g/L and the DHA yield was 5.8 g/L-11 g/L. The fatty acid contentwas 42.4%-53% by weight; the EPA content was 9.8%-22% of FAME; and theDHA content was 38.1%-43.6% of FAME. The lipid productivity was 1.9g/L/day-3.2 g/L/day, and the omega-3 productivity was 1.2 g/L/day-1.7g/L/day under these conditions, with 0.31 g/L/day-0.41 g/L/day EPAproductivity and 0.72 g/L/day-1.4 g/L/day DHA productivity.

Example 2 Apparent Digestibility of DHA and EPA as Microbial Biomass inAtlantic Salmon

Atlantic salmon of initial body weight ca 200 g were randomlydistributed into 1.5 m tanks with 50 fish per tank. The watertemperature was in the range of 10° C. Fish were adapted to the controldiet for 2 weeks before starting feeding the experimental diets.

A 3-mm diet was produced by extrusion at Nofima in Bergen according tothe formulation described in table 4. Only rapeseed oil was added to thediet and the basal levels of DHA and EPA in the control diet comes fromthe fish meal which has been included in the diet at a level of ca. 23%.NO fish oil was added to the diet.

As microbial source of DHA and EPA the biomass of a species ofSchizochytrium sp. ATCC PTA-10208 also known as OvegaGold™ has beenused. The biomass inclusions were 0, 0.9, 3.5 and 6.2% of the diet whichcorresponds to DHA levels of 0, 0.5, 1.0, 1.5%.

Fish were fed for at least 4 weeks and each dietary treatment wasperformed in triplicates. After 4 weeks of experimental feeding, feceswere collected by stripping each fish from each individual tank. Musclesample was taken out from five fish per tank.

Apparent digestibility coefficients were determined for the omega-3fatty acids and for the nutrients such as dry matter, lipids andprotein.

Statistical analysis was performed using Statbox Pro (one-way ANOVA).

TABLE 4 Diet formulation Major diet OvegaGold OvegaGold OvegaGoldingredients providing providing providing (%) Ctrl 0.5% DHA 1.0% DHA1.5% DHA Fish meal 23 22 22.4 22 Soy protein conc. 17 17 17 17 Rapeseedoil 22 21 20 19 Wheat meal 11.4 10.9 8.7 7.4 Corn gluten 5 5 5 5 Wheatgluten 13 13 13 13 Pea protein 2 2 2 2 concentrate OvegaGold 0 2.5 5.28.0

Results of in-feed recovery of DHA and EPA are presented in Table 5.Results show a very good recovery of DHA and EPA after feed processingby extrusion and a clear dose response.

TABLE 5 In feed recovery Recovery (mg/g) Treatments EPA DHA EPA + DHATotal Ω-3 Control 2.21 4.32 6.53 25.81 OvegaGold providing 4.31 9.6113.92 34.26 0.5% DHA From supplementation 2.1 5.3 6.4 OvegaGoldproviding 5.57 12.80 18.37 37.38 1.0% DHA From supplementation 3.4 8.511.9 OvegaGold providing 7.52 17.64 25.16 43.38 1.5% DHA Fromsupplementation 5.3 13.3 18.6

Apparent digestibility coefficient was determined for DHA, EPA, DHA+EPAand omega-3 fatty acids. FIGS. 1, 2 and 3 present the digestibly ofDHA+EPA, EPA and DHA, respectively. Results show that EPA and DHAprovided via an algal source are highly digestible.

FIG. 1: Apparent digestibility of DHA in the diet supplemented withgraded amounts of OvegaGold. Results of statistical analysis arepresented in Table 6.

FIG. 2: Apparent digestibility of EPA in the diet supplemented withgraded amounts of OvegaGold. Results of statistical analysis arepresented in Table 6.

FIG. 3: Apparent digestibility of DHA in the diet supplemented withgraded amounts of OvegaGold. Results of statistical analysis arepresented in Table 6.

FIG. 4: Muscle deposition of omega-3 fatty acids following ca 4 weeks offeeding the experimental diets. Muscle content confirms the highbioavailability of omega-3 fatty acids from a microalgal source.

TABLE 6 Apparent digestibility coefficients as mean ± Sd ADC (%) EPA +Total Treatment EPA ±SD DHA ±SD DHA ±SD Ω 3 ±SD Control 97.35 0.05 94.180.23 95.25 0.17 98.36 0.07 OvegaGold 98.50 0.06 96.96 0.06 97.40 0.0698.65 0.02 0.5% DHA OvegaGold 98.72 0.16 97.52 0.14 97.88 0.14 98.690.11 1.0% DHA OvegaGold 99.05 0.05 98.19 0.14 98.44 0.11 98.90 0.08 1.5%DHA

As a conclusion, DHA and EPA are highly bioavailable when provided as amicroalgal biomass supplemented to a salmon diet.

The invention claimed is:
 1. A method of sustainably producing anaquaculture meat product which comprises feeding an aquaculture feedcomposition to a fish over dietary cycles thereof, wherein all or partof fish oil in the aquaculture feed composition is replaced with asingle microbial source of eicosapentaenoic acid (“EPA”) anddocosahexaenoic acid (“DHA”), wherein the microbial source is microbialoil which is provided in a form selected from the group consisting ofpartially purified oil and purified oil, any of which is obtained fromthe microbial source, wherein the aquaculture feed composition comprisesa total amount of the EPA and DHA that is at least about 0.8%, measuredas a weight percent of the aquaculture feed composition, and wherein theEPA and DHA are present in a ratio of concentration of EPA toconcentration of DHA which is 1:1 or lower, based on individualconcentrations of the EPA and DHA in the microbial source or in theaquaculture feed composition.
 2. The method of claim 1, wherein themicroorganism from which the microbial source derives is an algae, fungior yeast.
 3. The method of claim 2, wherein the microorganism is amember of the genus Schizochytrium or Thraustochytrium.
 4. The method ofclaim 3, wherein the microorganism has the characteristics of thespecies deposited under ATCC Accession No. PTA-10208 or PTA-10209 orPTA-10210 or PTA-10211 or PTA-10212 or PTA-10213 or PTA-10214 orPTA-10215.
 5. The method of claim 3, wherein the microorganism is amutant strain of Schizochytrium.
 6. The method of claim 2, wherein themicroorganism is a transgenic microbe genetically engineered for theproduction of polyunsaturated fatty acid containing microbial oilcomprising EPA and DHA.
 7. The method of claim 1, wherein theaquaculture meat product has a ratio of concentration of EPA toconcentration of DHA that is equal to or lower than 1:1, based on theconcentration of each in the aquaculture meat product.
 8. An aquaculturefeed comprising: a single microbial source of eicosapentaenoic acid(“EPA”) and docosahexaenoic acid (“DHA”), wherein the microbial sourceis a microbial oil which is provided in a form selected from the groupconsisting of partially purified oil and purified oil, any of which isobtained from the microbial source, and wherein the EPA and DHA arepresent in the feed in an amount of at least about 0.8%, measured as aweight percent of the feed and in a ratio of concentration of EPA toconcentration of DHA which is equal to or less than 1:1, based onindividual concentrations of EPA and DHA in the microbial source or inthe feed.
 9. The aquaculture feed of claim 8, wherein the microorganismfrom which the microbial source derives is an algae, fungi or yeast. 10.The aquaculture feed of claim 9, wherein the microorganism is a memberof the genus Schizochytrium or Thraustochytrium.
 11. The aquaculturefeed of claim 10, wherein the microorganism has the characteristics ofthe species deposited under ATCC Accession No. PTA-10208 or PTA-10209 orPTA-10210 or PTA-10211 or PTA-10212 or PTA-10213 or PTA-10214 orPTA-10215.
 12. The aquaculture feed according to claim 10, wherein themicroorganism is a mutant strain of Schizochytrium.